Composition for testing for glucose in biological fluids



Patented Nov. 17, 1953 UNITED STATE KENT @FFKE Richard M. Carson andReuben R. Sacks, Dayton, Ohio No Drawing. Application January 23, 1952,Serial No. 267,937

2 Claims. I

This invention relates to a compositionand. method for testingbiological fluids to determine the presence of reducing substancestherein, is designed primarily for determining the presence of, and theamount of sugar in urine, and is in the nature of an improvement uponthe composition and method described in our application Serial No.288,668, filed. January 30, 1951, now Patent. No. 2,608,533.

This application is a continuation, in party of. our copen'dingapplication Serial No. 38,173, filed July 1948, now abandoned, forComposition and Method for. Testing Biological Fluids.

In the diagnosis and treatment of certain diseases it is essential toascertain whether the urine or blood contains sugar or alcohol and theamount thereof. This is particularly true in thetreatment for diabetes.No cure for that disease has been found but itcan usually be controlledby the regulation of the diet or by the administration of insulin, orboth. The character of the diet and/or the dosage of insulin aredetermined largely by the amount of sugar content in the urine, whichmay change from time to time. It is desirable therefore that the patientshould be able to test his urine periodically todetermine what if anychange in treatment is.

in the. color of the testing reagent and the amount. of sugar wasindicated by the particular shade. of resulting color, and wasdetermined by comparison with a color chart. Such a comparison by anuntrained eye is apt tobe inaccurate. and the color chart sometimesfades to an extent which is unnoticed by the patient but seriouslyaffects the determination. Some of the tests require the use ofexternalheat, which notonly produces highly objectionable odors,

but isinconvenient and subjects the patient todanger of burns. Many suchtests require considerable' time for their completion. The materials'required are usually expensive and in; some instances lack stability andquickly deteriorate,

Oneobjectof the present invention is to provide such a test which can beeasily and accurately eiiected by the ordinary patient with a minimumof, equipment.

A further object of the invention is to provide such a test which can beeffected without the use or" heat inany form.

A further object is to provide such a test which does not require acomparison of colors; which is both qualitative and quantitative; whichwill not be materially affected by other reducing subsances in the fluidbeing. tested, and which can be efiected. quickly and at a low cost.

A further object is to provide a reagent for efiecting such a test whichcan be supplied to the patient in a dry condition and prepared for, useby the mere addition of water in the proper amount and can therefore behandled, carried or transported without danger of spilling or leakage.

A further object of the invention is to provide a reagent which is muchless corrosive than are reagents heretofore used for such tests, andwhich can be easily protected against deterioration.

A further object of the invention is to provide a reagent which willreact with the reducing substances in the fluid being tested to impartto the testing solution a color which. will be the same regardless ofthe quantity of reducing substance in said fluid, and the time requiredfor the completion of the reaction will accurately indicate the quantityofv the reducing substance present in the fluid being tested.

A further object of the invention is to provide a method whereby such areagent can be easily and accurately produced.

A further object of the invention is to provide a test unit suitable fora single test which can be easily carried by the patient and discardedafter being used.

Other objects of the invention may appear as the method and apparatusare described in detail.

Our invention contemplates the provision. of a dry reagent containing acolor indicator, a stabilizing agent, a heat absorbing agent and, ifdesired; a color reinforcing agent. The reagent is in dry form and canbe packed, shipped and.

stored for relatively long. periods of time in this condition. Whenit isdesired to use the reagent a relatively small quantity may be placedsolution and mixed withv the biological iiuidto be tested or, undercertain circumstances, may be used in dry form.

The color indicator should be readily soluble in water, should impartdefinite color to even a relatively dilute solution and should reactreadily with a reducing agent to bring about a definite and noticeablechange in color. For this purpose we have found that the desired resultsare obtained where we employ as the color indicator, a manganate of analkali metal such as lithium, sodium, potassium, rubidium and cesium.Potassium or sodium manganate are more readily available at an economiccost and we prefer to employ potassium manganate because it isanhydrous.

To stabilize the alkali metal manganate we employ a stabilizing agentwhich should be alkali in nature, should be readily soluble in water,should not react with the manganate or inhibit or interfere with thereaction of the manganate with the reducing agent. These functions areserved by an alkali metal hydroxide. However, the alkali shouldpreferably be in the form which is not too caustic to handle byinexperienced operators and which generates relatively less heat ofsolution while still serving to stabilize the alkali metal manganatewhen in powdered form. For this purpose sodium aluminate and potassiumaluminate serve very satisfactorily and we prefer to use sodiumaluminatebecause it dissolves more readily.

The heat absorbing agent should have a negative heat of solution. Itshould be readily soluble in water and should not react with the otheringredients or inhibit or interfere with the reaction between the alkalimetal manganate and the reducing agent in the biological fluid. For thispurpose we have found that sodium nitrate and potassium nitrate servesatisfactorily and optimum results are obtained from sodium nitrate.

As the color reinforcing agent, we employ an agent which is red in thepresence of an alkali, which will not react with the other ingredientsor inhibit or interfere with the reaction between the alkali metalmanganate and the reducing agent in the biological fluid. For thispurpose we may employ a small trace of iron oxide, phenol red orbromcresol purple.

When our preparation is used for testing the presence or quantity ofreducing agents such as glucose in a biological fluid it is preferably(but not necessarily) dissolved in water. The proportions of ingredientsshould be such that the alkali metal manganate in the aqueous solutionis between approximately 04% and .4% by weight of the solution and thequantity of sodium or potassium aluminate is between approximately 2%and 6% by weight of the solution. This means that the caustic or alkalimetal hydroxide in the aluminate should be roughly between 1% and 3% ofthe test solution. In order to obtain these desired proportions in thetest solution the alkali metal manganate in the powdered preparationshould be between approximately .66% and 20% of the potassium or sodiumaluminate (i. e. between 1.3% and 40% of the alkali metal hydroxide inthe aluminate).

The quantity of sodium or potassium nitrate may be varied but it isdesirable to use the maximum amount of nitrate up to the limits of itssolubility. The quantity of sodium or potassium nitrate should be atleast two times the quantity (by weight) of the sodium or potassiumaluminate (i. e. approximately four times the caustic content of thealuminate). Optimum results are obtained by employing six or Seven t m sthe 4 quantity (by weight) of nitrate than aluminate (i. e.approximately thirteen times the caustic content of the aluminate) Onlya trace of the color reinforcing agent should be employed. Thus, thequantity should be such as not to disturb the normally green coloring ofthe preparation in its original form resulting from the color of thealkali metal manganate. However, this will be sufficient to reinforcethe color of the manganese dioxide which is precipitated when themanganate reacts with a reducing agent in the biological fluid.

The several components of the composition may be prepared in any desiredmanner and then thoroughly mixed together in finely divided form in theproportions indicated above. However, We have found that the reagent maybe conveniently produced by preparing a strong solution of an alkalimetal hydroxide, preferably sodium or potassium hydroxide, and aluminumhydroxide in water, thereafter heating the mixture to a fused mass andwhile the fused mass is still hot adding an alkali metal permanganatewhich is reduced to an alkali metal manganate. Thereafter, sodium orpotassium nitrate and a trace of the color reinforcing agent or pigmentis added to the mixture and the entire mass is finely comminuted andground to a fine powder which is readily soluble in water. The materialsare added in the proper quantities so as to produce a reagent having theingredients in the proportions indicated above. Specific examples ofreagents embodying our invention and the method of preparing them are asfollows:

Example 1 Approximately 5 grams of sodium hydroxide are mixed withapproximately 10 grams of aluminum hydroxide and approximately 5 cc. ofwater is added thereto. This concentrated solution is heated to form afused mass of sodium aluminate and while the fused mass is hot there isadded thereto approximately .3 gram of potassium permanganate which isreduced to potassium manganate in the mixture. Thereafter, approximatelygrams of sodium nitrate is added to the mixture and the entire mass isfinely comminuted, thoroughly mixed, ground to a fine powder. Thereagent thus prepared is strongly oxidizing in character, is green incolor, will readily dissolve in water and will react with a reducingsubstance in the fluid to be tested to change its color to a reddishbrown.

Example 2 Approximately 10 grams of sodium hydroxide are mixed withapproximately 10 grams of aluminum hydroxide and approximately '7 cc. ofwater is added thereto. This concentrated solution is heated to form afused mass of sodium aluminate and while the fused mass is hot there isadded thereto approximately .2 gram of potassium permanganate which isreduced to potassium manganate in the mixture. Thereafter approximatelygrams of sodium nitrate. .1 gram of iron oxide, and .005 gram of phenolred are added to the mixture and the entire mass is finely comminuted,thoroughly mixed and ground to a fine powder. The reagent thus preparedis strongly oxidizing in character, is green in color, the active agentswill readily dissolve in water and will react with a reducing substancein the fluid to be tested to change its color to a reddish brown,

Approximatelygrams of potassium hydroxide are mixed with approximately10 grams, or aluminum hydroxide and approximately '7' cc. of water isadded thereto. This concentrated solution is heated to form a fused massof potassium aluminate and while the fused mass is, hot there is addedthereto approximately .5'. gram of potassium permanganate whichvisreduced to potassium manganate in the mixture. Thereafter,approximatelyl'dO grams of sodium nitrate, .05 gram of iron oxide, and.005 gram of phenol red are added to the mixture and the entire mass isfinely comminuted, thoroughly mixed andlg fmmd' to a fine powder. Thereagent thus prepared, is, strongly oxidizing in character, i green incolor, the active agentsv will readily dissolve water and will reactwith a, reducing substance in the fluid to be tested to change its.color to a reddish brown.

Examp e 4 Approximately 10 grams, of sodium hydroxide. are'mixed withapproximately 10 grams of alui'nr num hydroxide and approximately 7 cc.of waterv is added thereto. This concentrated solution is heated to forma fused mass of sodium aluminate and while the fused mass is hot thereis added thereto approximately 3 grams of sodium permanganate which isreduced to sodium manganate o in the mixture. Thereafter, approximately50 grams of potassium nitrate, .15 gram of iron oxide, and .003 gram ofphenol red are added to the mixture and the entire mass is finelycomminuted, thoroughly mixed and ground to a fine powder. The reagentthus prepared is strongly oxidizing in character, is green in color, theactive agents will readily dissolve in water and will react with areducing substance in the fluid to be tested to change its color to areddish brown.

In the foregoing examples the permanganate of any of the other alkalimetals or mixtures thereof may be substituted for the potassiumpermanganate and the sodium permanganate.

A reagent embodying our present invention and made in accordance withany of the foregoing examples is particularly useful in determining thepresence of and the relative amount of sugar in urine. The method oftesting may vary but we prefer to dissolve the reagent in Waterimmediately prior to conducting the tests.

A small quantity of the reagent is dissolved in water in an amountsufiicient to form a solution of the desired strength and a small sampleof urine or other fluid to be tested is introduced into the solution.The sugar which, in the form of glucose, is present in varying amountsin diabetic urine is a strong reducing substance which reacts with theoxidizing agent to produce manganese dioxide, which is a precipitatereddish brown in color and changes the color of the solution from theoriginal green to an easily distinguishable reddish brown. In making thetests the proportions of urine and reagent are determined in part by thespeed with which the reaction is to be effected. While the speed of thereaction may be varied it is quickly completed but is not instantaneousand the time interval varies with the amount of sugar in the urine, thetime required for completion of the reaction decreasing as the amount ofsugar increases. Thus, the amount of sugar present in the urine isindicated by the time required for the reaction to take place, and

the completion thereof is indicated by the change in color of thesolution.

malslngthetest-a predeterminedamount of thereosent. suchv as a. measur damount. of nowderecl ea t. or tabl t containing a known amoun oiithreagent. is placedin a cl an. dry. transpar nt recep acl of, a sui ablsize. and shape, eferably a ial appr ximately-1 /8 inches lone and at;in h in iameter. A measured amount of water is added to the reagent,which s etely di solved. When this solution has been completed a,predetermined quantity, or sample, of urine addedto, the solution. andthe 311 8 91 the solution to chan e. from the green color to areddish-brown is noted.

proportion of urine. to reagent may vary in accordance-with thecharacter of the reagent or'thc desired speed of reaction. With, areagent havin the mposition above. set forth in Ex ample i it ispreferable to place 0.125 gram of the reagent in; the. vial and addthereto one cubic centimeter, 15, drops, of water. The vial is preferably shaken. to expedite solution. One drop of urine is then added,to: the solution and thoroughly m xed therewith, as by shaking. Thetime which lapses between the introduction of theurine into the solutionand the complete change or color is measured. When. a complete change ofcolor takes place in twenty seconds or less the rating is. four plus. Ifa complete change of color re-- quires. more than eighty seconds theamount of sugar is not of material importance and the result. isconsidered negative. The time intervals corresponding to the several.readings are shown in the following table, which is furnished to theatient:

Time 'Quantity'ol Glucose Present Oto 20 sec 4plus. n... 2% or more. 20to 40 sec 40 to 60 sec. 60 to sec Over 80 sec Should the test show arating of four plus and it is desired to determine the actual percentageof sugar present, a second test may be made by diluting a sample ofurine with an equal amount of water and using the same amount ofreagent. The reaction is timed in the same manner as above and theresult is multiplied by two.

While the reaction of the reagent on the sugar is sufficiently slow topermit proper timing it is nevertheless completed in less time than isrequired to oxidize other reducing substances contained in the urine andtherefore such other substances do not interfere with the test.

The foregoing test for determining the presence and the quantity ofsugar in urine contemplates using a reagent made in accordance withExample 1. It will be appreciated that reagents with varying proportionsof alkali metal manganate and alkali metal aluminate within the limitsset forth above may also be employed and that the time of reaction willvary as the proportions of ingredients are varied. Thus, if theproportion of either the aluminate or the manganate are increased withinthe indicated proportions, the reaction time is increased and viceversa. The reaction times of reagents of varying formulae may be readilydetermined by simple tests with a given quantity of urine having knownpercentages of sugar. It will also be understood that the reaction timewill be changed by varying the relative amount of urine used in thetests. Thus, where a relatively larger quantity of urine is employed thereaction time will be speeded up.

When the test is to be performed with a dry reagent about 0.25 gram ofpowdered reagent is placed on a waterproof surface, such as a piece ofglass, and one drop of urine i added to the powder, The same colorchange takes place as above if sugar is present and the change is almostinstantaneous and too rapid in time for quantitative use.

To further simplify the use of the test by the patient the reagent maybe supplied to the patient sealed in a transparent container, such asthe vial above mentioned, there being in each vial the quantity ofreagent required for a single test. When the test is to be made thepatient opens the vial, as by removing a sealing closure, and adds therequired water, in the amount above mentioned, and when the reagent iscompletely dissolved adds the sample of urine and times the reaction asabove described. Such a test unit, or several such test units, can beeasil carried by the patient either in his pocket or in a handbag so asto be available for use when needed. The only thing required other thanthe unit itself is a small liquid dropper.

From the foregoing, it will be appreciated that our test and our testingreagent is very simple in character. Its technique is easily acquired,

no knowledge of chemical reactions is required and it is not necessaryto compare various shades of colors with a color chart. Therefore, it iswell suited for use by patients. Furthermore, the test requires verylittle equipment, no external heat and only one reagent. It is effectedat room temperatures and produces no objectionable odors. Thus, it isconvenient for use in privacy in the home or when traveling. Moreover,the

cost per test is low.

While we have described our reagent and the preferred methods ofproducing and using the same we wish it to be understood that we do notdesire to be limited to the details thereof as various modifications mayoccur to a person skilled in the art.

We claim:

1. A method of preparing a reagent for testing biological fluids whichcomprises mixing 5 grams of sodium hydroxide with 10 grams of aluminumhydroxide, adding thereto 5 cc. of water, heating the mixture to a fusedmass to form sodium aluminate, adding to the fused mass While hot 0.3gram of potassium permanganate, adding approximately 80 grams of sodiumnitrate, and comminuting the entire mixture.

2. A dry reagent for determining the presence and proportion of glucosein biological fluids by a marked change of color of the reagent withinpredetermined periods of time and without the application of externalheat comprising a dry alkali metal manganate and a dry soluble alkalistabilizer selected from the group consisting of sodium aluminate andpotassium aluminate, the alkali metal manganate being in the proportionof between approximately .66% and 20% by Weight of the alkalistabilizer.

UNITED STATES PATENTS Name Date Carson Aug. 26, 1952 Number

2. A DRY REAGENT FOR DETERMINING THE PRESENCE AND PROPORTION OF GLUCOSEIN BIOLOGICAL FLUIDS BY A MARKED CHANGE OF COLOR OF THE REAGENT WITHINPREDETERMINED PERIODS OF TIME AND WITHOUT THE APPLICATION OF EXTERNALHEAT COMPRISING A DRY ALKALI METAL MANGANATE AND A DRY SOLUBLE ALKALISTABILIZER SELECTED FROM THE GROUP CONSISTING OF SODIUM ALUMINATE ANDPOTASSIUM ALUMINATE, THE ALKALI METAL MANGANATE BEING IN THE PROPORTIONOF BETWEEN APPROXIMATELY .66% AND 20% BY WEIGHT OF THE ALKALISTABILIZER.